1. Field of the Invention
Phytophthora is a fungal plant pathogen which causes devastating diseases of potatoes, including late-blight disease. This disease occurs in potato plants in the field as well as in storage. Fungicides have been utilized to control the pathogen, however, new strains have become resistant, resulting in extensive crop loss. This invention relates to novel primers which can be used to detect pathogenic Phytophthora species and to distinguish among the pathogenic species.
2. Description of the Relevant Art
Potato late blight caused by Phytophthora infestans (P. infestans) has become extremely severe in recent years due to the influx of a new more variable pathogen population. Extensive crop loss from late blight disease has occurred in many areas of the U.S. and Canada with the occurrance of both foliar destruction and the rotting of blighted tubers in storage. Often, tubers which are asymptomatic but contain latent P. infestans infection are placed in storage and are eventually partially or totally lost as infections develop and are exacerbated by secondary infection with other pathogens such as Erwinia carotovora subsp. carotovora (bacterial soft rot). A rapid, accurate test is thus needed to identify Phytophthora species in the field, in harvested tubers and in seedlots to determine levels of infection and to detect the pathogen when no visible symptoms occur. This enables growers to decide whether to harvest a crop or to declare it a loss. Results of such a test may also dictate the control measures a grower may invoke early in the season to control late blight, as well as provide confirmatory data about the presence of pathogens in seedlots which could affect certification and/or sale. Such a test is also needed to identify the specific organisms present in the tubers, i.e. to differentiate late blight from other tuber diseases such as pink rot and Pythium leak.
Microbiological and immunological assays as well as visual examinations have conventionally been utilized for detecting the presence of Phytophthora species in infested samples. However, these methods have suffered from an inability to clearly distinguish one species from another. For example, potato late blight infection can easily be confused with pink rot, and misdiagnoses may occur which could cause improper acceptance or rejection of seedlots during seed certification.
Various molecular approaches have been used to differentiate Phytophthora species, including use of random genomic fragments (Goodwin et al. 1989. Phytopathology. vol. 79, pp. 716-721; Goodwin et al. 1990. Appl. Environ. Micro. vol. 56, pp. 669-674), mitochondrial DNA (Forster et al. 1988. Mycologia. vol. 80, pp. 466-478; Forster et al. 1990. Exper. Mycol. vol. 14, pp. 18-31) and ribosomal DNA (Lee and Taylor. 1992. Mol. Biol. Evol. vol. 9, pp. 636-653; Lee et al. 1993. Phytopathology. vol. 83, pp. 177-181). For detection, tests based on the polymerase chain reaction (PCR) are the most rapid and sensitive available, and amplification by PCR for Phytophthora detection has been carried out by various investigators. Ersek et al. (1994. Applied and Environmental Microbiology. vol. 60, no. 7, pp. 2616-2621), for example, have reported amplification of DNA from P. parasitica and P. citrophthora with species-specific primers, and Niepold and Schober-Butin (1995. Microbiological Research. vol. 150, pp. 379-385) have disclosed primers derived from the repetitive sequence of P. infestans which were effective for detecting that species. In addition, a PCR-based detection method has been described for the raspberry root rot fungus P. fragariae var. rubi based on a highly repetitive fragment containing the small subunit ribosomal RNA gene (Stammler and Seemuller. 1993. Z. Pflanzen. Pflanzenschutz. vol. 100, pp. 394-400).
The search has continued, however, for additional primers which are useful in a rapid and sensitive assay for the detection of the pathogen and for distinguishing between species of Phytophthora which are potato pathogens.